Electrophoresis is the process of separating molecules on the basis of the molecule's migration in an electric field. In an electric field, a molecule will migrate towards the pole that carries a charge opposite to the charge carried by the molecule. The charge carried by a molecule depends upon the pH of the medium in which the molecule is migrating. A common electrophoretic procedure is to set up solutions at different pH at each end of an electric field. At a certain pH, the isoelectric point of a molecule is obtained and the molecule carries no net charge. Therefore, as the molecule crosses the pH gradient, the molecule reaches an isoelectric point and is immobile in the electric field. Therefore, this electrophoresis separates molecules according to their different isoelectric points.
Electrophoresis in a polymeric gel, such as a polyacrylamide gel or an agarose gel, adds two advantages to an electrophoretic system. First, the polymeric gel stabilizes the electrophoretic system against convective disturbances. Second, the polymeric gel provides a porous passageway through which the molecules must travel. Since larger molecules will travel more slowly through the passageways than smaller molecules, use of a polymeric gel permits the separation of molecules by both molecular size and isoelectric point.
Electrophoresis in a polymeric gel can also be used to separate molecules only by molecular size. Some groups of molecules, such as RNA and DNA molecules, all have the same isoelectric point. These groups of molecules will always migrate through an electric field across a polymeric gel on the basis of molecular size. Molecules with different isoelectric points, such as proteins, can be denatured in a solution of detergent, such as sodium dodecyl sulfate (SDS). The SDS-covered proteins will have similar isoelectric points and will migrate through the gel on the basis of molecular size. The separation of DNA molecules on the basis of their molecular size is an important step in determining the nucleotide sequence of a DNA molecule.
A polymeric gel electrophoresis system is typically set up in the following way: A gel-forming solution is allowed to polymerize between two glass plates that are held apart on two sides by spacers. These spacers determine the thickness of the gel. Typically, sample wells are formed by inserting a comb-shaped mold into the liquid between the glass plates at one end and allowing the liquid to polymerize around the mold. Alternatively, the gel may be cast with a flat top and a pointed comb inserted between the plates so that the points are slightly imbedded in the gel. Small, fluid-tight areas between the points can be filled with a sample.
The top and bottom of the polymerized gel are placed in electrical contact with two buffer reservoirs. Macromolecule samples are loaded into the sample wells. A sample-loading implement, such as a pipette, is inserted between the two glass plates and the sample is injected into the well. To prevent sample mixing, it is advantageous to inject the sample as close to the gel as possible. It is difficult to place the tip of the pipette or loading implement close to the gel because the pipette tip is often wider than the gel.
An electric field is set up across the gel, and the molecules begin to move into the gel and separate according to their size. The size-sorted molecules can be visualized in several ways. After electrophoresis, the gels can be bathed in a nucleotide-specific or protein-specific stain which renders the groups of size-sorted molecules visible to the eye. For greater resolution, the molecules can be radioactively labelled and the gel exposed to X-ray film. The developed X-ray film will indicate the migration positions of the labelled molecules.
Both vertical and horizontal assemblies are routinely used in gel electrophoresis. In a vertical apparatus, the sample wells are formed in the same plane as the gel and are loaded vertically. The wells can be as deep and wide as needed, but the thickness of the well is limited by the thickness of the gel.
Ultra-thin electrophoretic gels (less than 0.2 mm) are useful because they may be electrophoresed at a higher voltage than thicker gels. Therefore, the electrophoretic run is faster. Another advantage of ultra-thin gels is higher resolution because less sample is needed. Because of their thinness, the ultra-thin gels are fixed for autoradiography quickly and easily.
The problem of sample loading is especially burdensome with ultra-thin gel electrophoresis. The dimensions of the sample well are usually determined by the thickness of the gel. Therefore, ultra-thin gels have ultra-thin sample wells. Practically, it is difficult to load gels less than 1 mm thickness with a conventional pipette or less than 0.2 mm in thickness with a capillary tube. Sample loading can be accomplished using very thin, flat pipette tips or pulled glass capillaries to deposit samples into the wells. However, viscous samples are difficult to pipette with these loading devices because the devices can clog and break readily.
What is needed in the art of electrophoresis is a device that enables easier loading of ultra-thin electrophoretic gels.